α-Ketoglutarate stimulates pendrin-dependent Cl- absorption in the mouse CCD through protein kinase C

alpha-Ketoglutarate (alpha-KG) is a citric acid cycle intermediate and a glutamine catabolism product. It is also the natural ligand of 2-oxoglutarate receptor 1 (OXGR1), a G(q) protein-coupled receptor expressed on the apical membrane of intercalated cells. In the cortical collecting duct (CCD), Cl-/HCO3- exchange increases upon alpha-KG binding to the OXGR1. To determine the signaling pathway(s) by which alpha-KG stimulates Cl- absorption, we examined alpha-KG-stimulated Cl- absorption in isolated perfused mouse CCDs. alpha-KG increased electroneutral Cl- absorption in CCDs from wild-type mice but had no effect on Cl- absorption in pendrin knockout mice. Because Gq protein-coupled receptors activate PKC, we hypothesized that alpha-KG stimulates Cl- absorption through PKC. If so, PKC agonists should mimic, whereas PKC inhibitors should abolish, alpha-KG-stimulated Cl- absorption. Like alpha-KG, PKC agonist (phorbol-12,13-dibutyrate, 500 nM) application increased Cl- absorption in wild-type but not in pendrin null CCDs. Moreover, PKC inhibitors (2.5 mM GF109203X and 20 nM calphostin C), Ca2+ chelators (BAPTA, 10-20 mu M), or PKC-alpha or -delta gene ablation eliminated alpha-KG-stimulated Cl- absorption. We have shown that STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) gene ablation increases urinary alpha-KG excretion, renal pendrin abundance, and CCD Cl- absorption. However, in SPAK null CCDs, Cl- absorption was not activated further by luminal alpha-KG application nor was Cl- absorption reduced with the PKC inhibitor GF109203. Thus SPAK gene ablation likely acts through a PKC-independent pathway to produce a chronic adaptive increase in pendrin function. In conclusion, alpha-KG stimulates pendrin-dependent Cl-/HCO3- exchange through a mechanism dependent on PKC and Ca2+ that involves PKC-alpha and PKC-delta.